1. Field of the Invention
The present invention relates to a multimodal bioimaging, more particularly, to a method for cell labeling and imaging using perfluorocarbon (PFC) nanoemulsion containing optical nanoparticle.
2. Description of the Related Art
Labeling cells with nanoparticle having optical and magnetic properties is widely used in the area of cell imaging and disease treatment. The technology of internalizing nanoparticles into the cells is critical for cell labeling. The interaction between nanoparticle and cells depend on the cell surface characteristics, therefore, the technology for reforming the surface of nanoparticle is very important in cell labeling. In general, internalizing functional nanoparticles into cells is performed by coating the nanoparticle surface with signaling peptides that are related to transfection, which can induce an interaction between the cells and the nanoparticle. Phagocytic cells, such as macrophages show high interaction with nanoparticles without any specific surface modifications, however there is difficulty in transporting nanoparticles into non-phagocytic cells, such as stem cells and NK cells. For non-phagocytic cell labeling, electroporation or nucleofection techniques, which transport nanoparticle or genes into cells using mechanical forces, are widely used. The technology for labeling cells with imaging agent is critical when monitoring the cells in the field of using stem cells and immune cells for treating diseases, such as cancers.
Hematopoietic stem cells are one of adult human stem cells with the ability to differentiate into blood forming cells (red blood cell, white blood cell, platelets and lymphocytes). Hematopoietic cells in the bone marrow continuously self-renew into cells forming the immune system. One of the cells of the immune system is Natural Killer cell (NK cell), which can nonspecifically kill cancer cells. The cytotoxicity of NK cell, such as lymphokine activated killer cell (LAK) and tumor infiltration lymphocytes (TIL) can be used to treat solid tumor or used for immune therapy by donor lymphocyte infusion method (Itoh, K, et al., J. Immunol., 36: 3910-3915, 1986; Bordignon C. et al., Haematologica, 84: 1110-1149, 1999) which can be used as a new cell treatment method to prevent the immune rejection response after stem cell transplant or organ transplant. In addition, depletion of NK cell differentiation and activity have been reported to be involved in various disease such as breast cancer (Konjevi G, et al., Breast Cancer Res. Treat., 66: 255-263, 2001), subcutaneous melanoma (Ryuke Y, et al., Melanoma Res., 13: 349-356, 2003) and lung cancer (Villegas F R, et al., Lung Cancer, 35: 23-8, 2002), therefore suggesting a possible NK cell treatment for treating these diseases.
However, due to its difficulty in introducing image contrast reagent into NK cells or labeling the NK cells, there is no report on using a molecular imaging agent for imaging or labeling NK cells to treat disease (Giovanni Lucignani et al., Trends Biotechnol., 24: 410-418, 2006). Recently, a method of introducing image contrast reagents into genetically modified NK cells expressing HER-2/neu receptor using lipofection and electroporation method (Eur Radiol., 15: 4-13, 2005) and an indirect treatment method by expressing reporter gene using retroviral vector (Edinger, M. et al., Blood, 101: 640-648, 2003) has been introduced, but had the disadvantage of affecting the cell activity.
Perfluorocarbon (PFC) is widely used in various areas in industry due to its low viscosity, low dielectric constant, high vapor pressure, high compressibility and high gas solubility. When applied to clinical use, perfluorocarbon has an advantage of having high density, antifriction properties and magnetic susceptibility values close to water. In addition, high dose of perfluorocarbon is safe in human body and pure fluorocarbon within certain molecular weight range (460-520 Da) is non-toxic, non-carcinogenic, non mutagenic and non-teratogenic and does not trigger immune responses. Perfluorocarbon is widely applied in liquid ventilation, oxygen delivery and imaging due to its physiological inactivity and biocompatibility (Marie Pierre Krafft, Advanced Drug Delivery Reviews, 47:209, 2001; Gregory M., et al., Current Topics in Developmental Biology, 70:57, 2005).
The gas phase PFC which forms lipid coated low density micro-bubbles have been widely studied as an ultrasound imaging agent and are being used commercially. PFC micro-bubbles have been studied as transporting bioactive materials to specific local regions and for the treatment purpose due to its cavitation by ultrasound energy. The study is focused in diagnostic imaging of cardiovascular related cancers and improving treatment by drug delivery (Evan C et al., Advanced drug delivery Reviews, 56: 1291, 2004).
Perfluorocarbon (PFC) is an excellent contrast material for MRI, the PFC and derivatives to have been actively studied in 19F MRI fields. As compared with conventional 1H MRI imaging agents, 19F has an almost identical gyromagnetic ratio to protons, a spin ½ nucleus and 100% of natural isotope abundance. It has an advantage of not being a source of a man-made pollution and has a background concentration which is same as the environmental concentration found in nature.
PFC emulsion nanoparticle may be functionalized as a MRI molecular imaging agents by bonding paramagnetic chelates and homing ligands onto an external phospholipid monolayer in MRI field, and many studies of the PFC emulsion nanoparticles have been conducted as drug delivery vectors including bioactive agents (US 2004/0115192 A1; U.S. Pat. No. 6,676,963 B1; US 2003/0086867; US 2003/0215392 A1; US 2004/0248856 A1). In the MRI, the nuclei are dephased and then rearranged in the direction of a magnetic field. At this time, the process of supplying energy to the lattice of the nuclei to reach a thermal equilibrium is referred to as T1. The PFC emulsion nanoparticles are used as T1-weighted ultraparamagnetic imaging agents reflected in particulate or molecular relaxivity. For the imaging agents to have maximum relaxivity, all the paramagnetic materials used as the imaging agents are necessarily in an external aqueous phase. In 1.5 T, the molecular relaxivity of the PFC nanoparticles depends on lipophilic chelation and has a value of 1,000,000 to 2,000,000 mMs−1 (Flacke et al, Circulation, 104:1280, 2001; Winter et al, Magn. Reson. med., 50:411, 2003).
Biotin ligand sandwiched with biotin treated nanoparticle by streptavidin-biotin interaction for molecular imaging, fibrin imaging which can detect the blood clots by direct covalent bonding of ligand and the monoclonal antibody targeting of molecular epitopes, this research was performed on imaging of angiogenesis in cancer and imaging of early atherosclerosis to detect the low expression level of αvβ3 integrin and for anti-angiogenesis treatment (U.S. Pat. No. 5,690,907; US 2004/0058951 A1; US 2006/0147380 A1).
The PFC emulsion nanoparticle currently used for molecular imaging are using the MR characteristic of 19F or loading contrast reagent for 1H MR by adding a reagent that can chelate gadolinium (Gd) that can be used as the T1 contrast agent that envelops the lipid layer of PFC emulsion nanoparticle.
The present inventors have discovered that perfluorocarbon nanoemulsion containing optical nanoparticle can label various cell types from phagocytic cells that are readily labeled, to NK cells that are difficult to be labeled. The labeling exerted no effect on cell viability and function of the NK cells. As a result, the inventors have developed multifunctional cell labeling and imaging techniques that can be used in magnetic resonance imaging (MRI) and optical imaging (OI) simultaneously, thus completed the present invention.